The reaction between glucose and proteins has been known for some time. Its earliest manifestation was in the appearance of brown pigments during the cooking of food. In 1912, Maillard observed that glucose or other reducing sugars react with amino acids to form adducts that undergo a series of dehydrations and rearrangements to form stable brown pigments (Maillard, 1912, C. R. Acad. Sci. 54:66-68).
Thus, the nonenzymatic reaction between glucose and the free amino groups on proteins to form a stable amino, 1-deoxy ketosyl adduct, known as the Amadori product, has been shown to occur with hemoglobin, wherein the reaction of glucose with the amino terminus of the .beta.-chain of hemoglobin forms the adduct known as hemoglobin A.sub.1c. The reaction has also been found to occur with a variety of other body proteins, such as lens crystallin, collagen and nerve proteins (see Bunn et al., 1975, Biochem. Biophys. Res. Commun. 67:103-109; Koenig et al., 1975, J. Biol. Chem. 252:2992-2997; Monnier and Cerami, in Maillard Reaction in Food and Nutrition, ed. Waller, G. A., American Chemical Society 1983, pp. 431-448; and Monnier and Cerami, 1982, Clinics in Endocrinology and Metabolism 11:431-452).
Moreover, brown pigments with spectral and fluorescent properties similar to those of late-stage Maillard products have also been observed in vivo in association with several long-lived proteins, such as lens proteins and collagen from aged individuals. An age-related linear increase in pigment was observed in human dura collagen between the ages of 20 to 90 years (see Monnier and Cerami, 1981, Science 211:491-493; Monnier and Cerami, 1983, Biochem. Biophys. Acta 60:97-103; and Monnier et al., 1984, Proc. Natl. Acad. Sci. USA 81:583-587).
Glucose and other reducing sugars attach non-enzymatically to the amino groups of proteins in a concentration-dependent manner. Over time, these initial Amadori adducts can undergo further rearrangements, dehydrations and cross-linking with other proteins to accumulate a family of complex structures referred to as Advanced Glycosylation Endproducts (AGEs). Substantial progress has been made toward the elucidation of the role and clinical significance of advanced glycosylation endproducts, so that it is now acknowledged that many of the conditions heretofore attributed to the aging process or to the pathological effects of diseases such as diabetes, are attributable at least in part to the formation of AGEs in vivo.
AGE accumulation can be indicative of protein half-life, sugar concentration, or both. These factors have important consequences. Numerous studies have indicated that AGEs play an important role in the structural and functional alterations which occur during aging and in chronic disease. Additionally, advanced glycosylation endproducts are noted to form more rapidly in diabetic and other diseased tissue than in normal tissue.
Hb-AGE has been found to be predictive of aging or disease progression over the long term (International Patent Publication No. WO 93/13421 by Bucala, published Jul. 8, 1993; Makita et at., 1992, Science 258:651-653). Hb-AGE measurements provide an appropriate index of long-term tissue modification by AGEs and are useful in assessing the contribution of advanced glycosylation to a variety of diabetic and age-related complications. While hemoglobin A.sub.1c (HbA.sub.1c) has been reported as predictive of the extent of glycation on the hemoglobin .beta. chain, HbA.sub.1c is only a reversible intermediate in the advanced glycosylation pathway and numerous other intermediates are believed to exist. Hb-AGE, as an irreversible adduct, is a superior measure of disease progression, drug effectiveness, etc.
Hb-AGEs are used to more readily correlate the progression of disease and longer term control of blood sugar levels. HbA.sub.1c is a reversible intermediate, and reaches equilibrium with glucose over a 3-4 week period. Hence, levels of HbA.sub.1c only reflect blood glucose only during this short time period. Hb-AGE, in contrast, is an irreversible adduct and reflects blood sugar over the lifespan of hemoglobin. Thus, the effectiveness of treatment for AGE-related diseases or disorders can also be determined from the level of Hb-AGE in samples. Thus, the reduction in Hb-AGE levels as a result of aminoguanidine therapy is a primary example of the successful pharmacological inhibition of advanced glycosylation in human subjects.
Prior to the instant invention, detection of Hb-AGEs required a complex assay format, that included TCA precipitation of the hemoglobin from the hemolysate, followed by centrifugation to separate the precipitated protein from the supernatant liquid fraction. Resolution of the precipitate was accomplished by adding sodium hydroxide at high pH (pH&gt;11), followed by pH adjustment with 0.3M KH.sub.2 PO.sub.4, pH 7.4 buffer (Makita et al., 1991, Diabetologia 34:40-45). After pH adjustment to about 7.8, some of the hemoglobin precipitates out of solution, thus requiring a separation step prior to assay of the liquid fraction. In short, the current protocol complex, is cumbersome, time consuming, subject to and generally not applicable to a clinical laboratory setting.
Accordingly, there is a need in the art for a simpler, faster, and milder sample treatment protocol to facilitate testing for Hb-AGE in clinical laboratory settings.
There is a further need in the art for kits containing the reagents necessary to perform such assays.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.